ISO 15685:2004

Soil quality — Determination of potential nitrification and inhibition of nitrification — Rapid test by ammonium oxidation

ISO 15685:2004 specifies a rapid method for the determination of the potential rate of ammonium oxidation and inhibition of nitrification in soils. This method is suitable for all soils containing a population of nitrifying microorganisms. It can be used as a rapid screening test for monitoring soil quality and quality of wastes, and is suitable for testing the effects of cultivation methods, chemical substances and pollution in soils.

Qualité du sol — Détermination de la nitrification potentielle et inhibition de la nitrification — Essai rapide par oxydation de l'ammonium

General Information

• Status: Withdrawn
• Publication Date: 29-Mar-2004
• Withdrawal Date: 29-Mar-2004
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: ISO/TC 190/SC 4 - Biological characterization
• Drafting Committee: ISO/TC 190/SC 4/WG 4 - Effects on soil micro-organisms
• Current Stage: 9599 - Withdrawal of International Standard
• Completion Date: 10-Jul-2012

Relations

• Revised: ISO 15685:2012 - Soil quality — Determination of potential nitrification and inhibition of nitrification — Rapid test by ammonium oxidation
• Effective Date: 10-Jan-2009

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 15685
First edition
2004-03-15
Soil quality — Determination of potential
nitrification and inhibition of
nitrification — Rapid test by ammonium
oxidation
Qualité du sol — Détermination de la nitrification potentielle et inhibition
de la nitrification — Essai rapide par oxydation de l'ammonium

Reference number
ISO 15685:2004(E)
©
ISO 2004

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ISO 15685:2004(E)
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ISO 15685:2004(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International
Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 15685 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
methods.
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iv

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INTERNATIONAL STANDARD ISO 15685:2004(E)
Soil quality — Determination of potential nitrification and
inhibition of nitrification — Rapid test by ammonium oxidation
1Scope
This International Standard specifies a rapid method for the determination of the potential rate of ammonium
oxidation and inhibition of nitrification in soils. This method is suitable for all soils containing a population of
nitrifying microorganisms. It can be used as a rapid screening test for monitoring soil quality and quality of
wastes, and is suitable for testing the effects of cultivation methods, chemical substances and pollution in soils.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced document
(including any amendments) applies.
ISO 11465:1993, Soil quality — Determination of dry matter and water content on a mass basis — Gravimetric
method
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
inhibitory dose
ID
amount of a chemical added to soil that effectively inhibits biological activity by a stated percentage after a given
time, in comparison to an untreated control
NOTE It is expressed as a percentage. For example, ID25 and ID50 indicate 25 % and 50 % inhibition of biological activity,
respectively.
3.2
biosolid
organic fertiliser used in agriculture, including sewage sludge, compost, manure and industrial products
4 Principle
Ammonium oxidation, the first step in autotrophic nitrification in soil, is used to assess the potential activity of
microbial nitrifying populations. Autotrophic ammonium-oxidizing bacteria are exposed to ammonium sulfate in
a soil slurry buffered at pH 7,2. Chemical test substances, extracts of biosolids, as well as samples of polluted
soils can be added to the slurry at various concentrations. Oxidation of the nitrite formed by nitrite-oxidizing
bacteria in the slurry is inhibited by the addition of sodium chlorate. The subsequent accumulation of nitrite is
measured over a 6-h incubation period, and is taken as an estimate of the potential activity of ammonium-
oxidizing bacteria. As the generation time of ammonia-oxidizing bacteria is long (> 10 h), the method provides
a measure of the potential activity of the nitrifying population at the time of sampling. It does not measure growth
of the nitrifying population.
Nitrite can be measured quickly and accurately and hence the method is fast, accurate, reproducible and
inexpensive.
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ISO 15685:2004(E)
Substances known to be active at pH< 7,2 should be added before starting the test in due time to allow
substances to exhibit their effects on nitrifying bacteria.
5Reagents
5.1 Distilled water.
5.2 Potassium dihydrogen phosphate, .c(KH PO )= 0,2 mol/l
2 4
5.3 Dipotassium hydrogen phosphate, .c(K HPO )= 0,2 mol/l
2 4
5.4 Sodium chlorate, .c(NaClO )= 0,5 mol/l
3
5.5 Diammonium sulfate, (NH ) SO
4 2 4.
5.6 Sodium hydrogen carbonate, .c(NaHCO )= 5 mmol/l
3
5.7 Potassium chloride, .c(KCl)= 4 mol/l
5.8 Stock solution A.
Prepare stock solution A by combining the following:
KH PO (5.2) 28 ml
2 4
K HPO (5.3) 72 ml
2 4
Distilled water (5.1) 100 ml
5.9 Test medium.
Prepare test medium by combining the following:
Stock solution A (5.8) 10 ml
Sodium chlorate, NaClO (5.4) 10 ml to 30 ml
3
Diammonium sulfate, (NH ) SO (5.5) 0,198 g
4 2 4
Dilute to 1 000 ml with distilled water (5.1).
The final medium contains 1 mmol/l of potassium phosphate buffer, 5 mmol/l to 15 mmol/l of sodium chlorate
1,5 mmol/l
and of diammonium sulfate, and has a pH of approximately 7,2.
The sodium chlorate concentration selected within the range given should be sufficient for effective inhibition of
biological nitrate formation, but at the same time not have negative effects on ammonium oxidation. If
necessary, test the influence of sodium chlorate concentration.
Test chemicals to be added to the test medium shall be dissolved in the phosphate buffer described and added
before diluting to 1l. For test chemicals with low water-solubility, prepare solutions by mechanical dispersion or
by use of vehicles such as organic solvents, emulsifiers or dispergents of low toxicity to ammonium-oxidizing
bacteria. The concentration of a vehicle should not exceed 100 mg/l in the final medium.
Add extracts of biosolids, when tested, separately to the test medium before dilution to 1l.
When a carbon source is needed, e.g. in testing of biosolids with high nitrate levels, add 10 ml of NaHCO (5.6)
3
to the test medium before diluting to 1l.
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ISO 15685:2004(E)
6 Apparatus
6.1 Ordinary laboratory equipment.
6.2 Orbital shaking incubator, thermostatically controlled.
7 Sampling, storage and characterization of samples
Guidance on sampling, sample processing and storage is given in ISO 10381-6. For testing of chemicals or
biosolids, or for mixing tests with polluted soils, experimental soil with ammonium-oxidizing activity of between
200 ng N/g dry mass/h to 800 ng N/g dry mass/h determined from preliminary experimentation should be
chosen.
◦
Samples of experimental soils can usually be stored in a refrigerator at approximately 4 C for periods of up to
three months prior to use (see ISO 10381-6). Some soils can only be kept in the refrigerator for up to two
◦
weeks. Storage of soil samples in a freezer (−20 C) is not generally recommended.
◦
It has been shown for a number of soils from temperate climates that storage at −20 C for up to 12 months
[3]
does not inhibit ammonium oxidation activity . Where such information is available for a particular soil, storage
under such conditions is permissible.
Measurement of the following variables is recommended for characterization of the soil (see Bibliography):
— particle size distribution (ISO 11277);
— water content (ISO 11465);
— water-holding capacity (ISO 14238:1997, Annex A);
— pH (ISO 10390);
— effective cation-exchange capacity (ISO 11260);
— organic matter content (ISO 10694);
— total nitrogen content (ISO 11261).
8 Procedure
8.1 Testing soils
8.1.1 General
The test design should include at least three replicates of approximately 25 g of moist soil. Determine
gravimetrically the water content of the soil for calculation of dry mass according to ISO 11465.
8.1.2 Initial incubation
Mix soil samples or waste materials with test medium (5.9) to form slurries. The volume of test medium should
be adjusted to give a precise total liquid volume, e.g. 100 ml. Calculate the volume of medium to be added by
subtracting the volume of water in the initial soil or waste sample from the desired liquid volume, e.g. .
100 ml
Incubate the slurries in 250 ml flasks standing upright on an orbital shaking incubator (6.1), thermostatically
◦ ◦
controlled to 25 C± 2 C. Rotation should be sufficient to keep solids suspended.
A volume of liquid greater than 100 ml is required in the slurry if the water-holding capacity of the soil is
> 200 % (organic soils).
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ISO 15685:2004(E)
8.1.3 Sampling of soil slurry
Take samples (2ml) of the soil slurry after 2h and 6h of incubation, provided that ammonium oxidation is
known to be linear over this period. The soil slurry should be well shaken at sampling times to ensure that the
ratio of solution/soil is constant during the test. Dispense samples into test tubes and add 2ml of KCl (5.7) to
stop the ammonium oxidation. Then centrifuge the samples at e.g. 3 000g for 2 min, or filter. Filter paper should
be of high filtration speed, while its chemical purity may be less than the highest grade
...